MiR-2 family targets <i>awd</i> and <i>fng</i> to regulate wing morphogenesis in <i>Bombyx mori</i>

MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. <i>miR-2</i> is an invertebrate-specific miRNA family that has been predicted in the fruit fly, <i>Drosophila melanogaster</i>, to be involved in regulating the Notch signaling pathway. We show here that <i>miR-2</i> plays a critical role in wing morphogenesis in the silkworm, <i>Bombyx mori</i>, a lepidopteran model insect. Transgenic over-expression of a <i>miR-2</i> cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that <i>miR-2</i> regulates functions essential for normal wing morphogenesis. Two genes, <i>abnormal wing disc</i> (<i>awd</i>) and <i>fringe</i> (<i>fng</i>), which are positive regulators in Notch signaling, are identified as <i>miR-2</i> targets and validated by a dual-luciferase reporter assay. The relative abundance of both <i>awd</i> and <i>fng</i> expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of <i>awd</i> and <i>fng</i> using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the <i>miR-2</i> overexpression transgenic animals. The critical role of <i>miR-2</i> in <i>Bombyx</i> wing morphogenesis may provide a potential target in future lepidopteran pest control.